Instability of orthophthalaldehyde reagent for amino acid analysis

Determination of amino acids by reversed-phase chromatography of the adduct with orthophthalaldehyde and a thiol is rapid and sensitive. The major recognized adverse feature of this method is the instability of the reaction product, which requires precise control of reaction timing and chromatographic parameters for reliable quantitative application. We report another source of major variability: reagent instability. Deterioration of reagent was noted as low peak heights and peak broadening and was predictable if the premixed reagent was left at room temperature. Restoration of sharp chromatograms was accomplished by addition of mercaptoethanol or sodium metabisulfite. Reagent which was chromatographically inert contained minimal free thiol by direct assay. Free thiol disappearance was markedly slowed by addition of a chelating agent. Excess mercaptoethanol was deleterious. We conclude that reagent deterioration represents oxidation of the thiol, may be reversed by rereduction with minimal thiol or bisulfite, and may be minimized by inclusion of a metal chelator in the reagent.     

Localization of the Miller-Dieker critical region is proximal to locus D17S34 (p144D6) in 17p13.3

A child with normal growth and development and the abnormal karyotype 46,XY,17ps, was analyzed using molecular probes localized to 17p13. The results indicated the presence of two copies of the probes YNZ22.1 (D17S5) and YNH37.3 (D17S28), previously shown to be deleted in all Miller-Dieker (MDS) patients studied. However, the patient was hemizygous for probe p144D6 (D17S34), which is absent in approximately 75% of the MDS patients. As the patient is active at 9 months of age, with no clinical signs of MDS, the results confirm that the absence of locus D17S34 does not lead to the phenotypic expression of MDS. Furthermore, this deletion should assist in defining the distal limits of this contiguous gene syndrome.     

Endogenous lipid pneumonia in opossums from Louisiana

Endogenous lipid pneumonia was present in 19 of 27 opossums (Didelphis virginiana) trapped in the vicinity of Baton Rouge, Louisiana. The severity of lesions varied from small pleural and subpleural aggregates of foamy macrophages with minimal disruption of pulmonary architecture to large nodular accumulations of foam cells with cholesterol clefts and localized emphysema. The cause of the lipid pneumonia may be related to pulmonary nematode parasite infections, which were evident in 13 of the affected animals.     

Studies on the Tissue Distribution of the Puromycin-Sensitive Enkephalin-Degrading Aminopeptidases

An antiserum generated to the soluble form of the rat brain puromycin-sensitive enkephalin-degrading aminopeptidase was used to determine the tissue distribution of the soluble and membrane-associated forms of this enzyme. All tissues examined contained significant levels of the soluble enzyme form, with this enzyme accounting for greater than 90% of the arylamidase activity in brain, heart, and skeletal muscle. Native gel electrophoresis coupled with activity staining as well as inhibition studies were used to confirm the presence of this enzyme in various tissues. Serum was found not to contain this particular aminopeptidase. In contrast to the results obtained with the soluble enzyme form, brain was the only tissue found to contain the membrane-associated enzyme form. Although all tissues contained membrane-associated aminopeptidase activity only the brain enzyme could be maintained in solution in the absence of detergent. In addition, the brain membrane-associated enzyme could be distinguished from the membrane-associated aminopeptidase activity in other tissues on the basis of its sensitivity to inhibition by puromycin.     

Analgesia induced by N-acetylserotonin in the central nervous system

The relationship between N-acetylserotonin (NAS) in the central nervous system (CNS) and responses to pain was investigated. Using the rat tail-flick model, we initially replicated the work of others showing that intraventricular (IVC) injection of a dipeptide structurally similar to both NAS and serotonin was capable of inducing analgesia in the rat. We then showed that IVC-NAS, but not serotonin elicited analgesia in much the same manner as the dipeptide. This effect proved to be very specific as it required the presence of both an acetyl group on the terminal side chain amine as well as a hydroxyl group on the C-5 position of the indole ring. Substitution of the C-5 hydroxyl by a methoxyl group (melatonin) abolished the analgesic effect. Similarly, removing the N-acetyl substitution (serotonin) also eliminated the analgesia. IVC injection of highly specific antiserum to NAS induced hyperalgesia. Furthermore, an interaction was found between NAS and opiate systems. We demonstrated that while naloxone, the opiate antagonist, has no hyperalgesic properties of itself, it did counteract the analgesia induced by NAS. Similarly, NAS antiserum reversed the analgesia induced by the opiate morphine. This work provides evidence that NAS is an endogenously active substance within the CNS pain network.     

Taxol-induced reorganization of the microtubule system in murine splenic lymphocytes inhibits response to allogeneic cells but not to concanavalin A

The exposure of mouse splenic lymphocytes to the microtubule assembly-promoting drug taxol (10 microM for 4 h) results in an extensive reorganization of the microtubule system to form one to a few large bundles of microtubules, which extend from the centrosome. Lymphocytes pretreated with taxol for 4 h, or cultured in the continued presence of taxol, respond normally to the mitogen concanavalin A up to, and including, the stage of DNA replication. In contrast, the induction of DNA synthesis during the alloactivation of lymphocytes is inhibited when taxol is present in the mixed leukocyte culture. If the stimulators are pretreated with this drug, the mixed leukocyte reaction occurs normally, but pretreatment of the responders inhibits the proliferative response markedly. Microscopic observations of nuclear morphologies in these populations and autoradiography indicate that taxol inhibition occurs early in alloactivation, prior to DNA replication. The responding ability of taxol-treated lymphocytes is not restored to control levels by the addition of interleukin 2, leading to the suggestion that interleukin 2 receptors do not emerge or function normally in these cells. We conclude that the capacity to respond to allogeneic cells, but not to a mitogen, is dependent on the presence of the normal submembranous organization of the microtubule system.     

The statistical analysis of circadian phase and amplitude in constant-routine core-temperature data.

Accurate estimation of the phases and amplitude of the endogenous circadian pacemaker from constant-routine core-temperature series is crucial for making inferences about the properties of the human biological clock from data collected under this protocol. This paper presents a set of statistical methods based on a harmonic-regression-plus-correlated-noise model for estimating the phases and the amplitude of the endogenous circadian pacemaker from constant-routine core-temperature data. The methods include a Bayesian Monte Carlo procedure for computing the uncertainty in these circadian functions. We illustrate the techniques with a detailed study of a single subject's core-temperature series and describe their relationship to other statistical methods for circadian data analysis. In our laboratory, these methods have been successfully used to analyze more than 300 constant routines and provide a highly reliable means of extracting phase and amplitude information from core-temperature data.